The emergence of protein complexes: quaternary structure, dynamics and allostery. Colworth Medal Lecture

All proteins require physical interactions with other proteins in order to perform their functions. Most of them oligomerize into homomers, and a vast majority of these homomers interact with other proteins, at least part of the time, forming transient or obligate heteromers. In the present paper, we review the structural, biophysical and evolutionary aspects of these protein interactions. We discuss how protein function and stability benefit from oligomerization, as well as evolutionary pathways by which oligomers emerge, mostly from the perspective of homomers. Finally, we emphasize the specificities of heteromeric complexes and their structure and evolution. We also discuss two analytical approaches increasingly being used to study protein structures as well as their interactions. First, we review the use of the biological networks and graph theory for analysis of protein interactions and structure. Secondly, we discuss recent advances in techniques for detecting correlated mutations, with the emphasis on their role in identifying pathways of allosteric communication.     

Inulinase immobilization on polyethylene glycol/polypyrrole multiwall carbon nanotubes producing a catalyst with enhanced thermal and operational stability

This paper describes the development of a simple method for mixed non‐covalent and covalent bonding of partially purified inulinase on functionalized multiwall carbon nanotubes (f‐MWCNTs) with polypyrrole (PPy). The pyrrole (Py) was electrochemically polymerized on MWCNTs in order to fabricate MWCNTs/PPy nanocomposite. Two multiple forms of enzyme were bound to N‐H functional groups from PPy and ‐COO^? from activated MWCNTs to yield a stable MWCNTs/PPy/PEG immobilized preparation with increased thermal stability. Fourier transform infrared (FTIR) spectroscopy and scanning electron microscopy (SEM) were used to confirm functionalization of nanoparticles and immobilization of the enzyme. The immobilization yield of 85% and optimal enzyme load of 345 μg protein onto MWCNTs was obtained. The optimum reaction conditions and kinetic parameters were established using the UV‐Vis analytical assay. The best functional performance for prepared heterogeneous catalyst has been observed at pH 3.6 and 10, and at the temperatures of 60 and 80ºC. The half‐life (t_1/2) of the immobilized inulinase at 60 and 80ºC was found to be 231 and 99 min, respectively. The reusability of the immobilized formulation was evaluated based on a method in which the enzyme retained 50% of its initial activity, which occurred after the eighteenth operation cycle.     

Plitvice Lakes National park harbors ancient,yet endangered diversity of trout (genus Salmo)

National park Plitvice Lakes is the oldest, the largest and the most visited national park in Croatia, well known for its stunning karstic watersheds that supply Plitvice Lakes and exceptionally valuable biodiversity. Lack of knowledge on the taxonomic status, population structure and viability of trout in the Plitvice Lakes National park, as well as the possibility of existence of cryptic diversity, motivated this study which aimed to determine: taxonomic status and phylogenetic relationship of Plitvice Lakes trout with other Salmo species, genetic structure and diversity of their populations, as well as future viability of populations. 150 samples of trout were obtained from 17 localities. Two mitochondrial gene markers, gene for cytochrome b and mitochondrial control region were used in phylogenetic reconstruction, as well as in population genetic investigation. Intraspecific and intrapopulation genetic diversity was described by calculating several measures of genetic polymorphism, while gene flow among populations was estimated as the migration rates and as the number of immigrants per generation. Phylogenetic trees revealed that Plitvice Lakes watershed is inhabited by two evolutionary independent units of the genus Salmo, representing two species, Salmo labrax Pallas, 1814 and Salmo trutta Linnaeus, 1758. Possibly ‘pure’ populations of both species were found alongside several admixed ones in which both species are present. Our data has shown low intraspecific and intrapopulational diversities for Plitvice Lakes trout and restricted gene flow. Small effective sizes of populations and weak interconnections between them, which are most likely a consequence of fragmentation and habitat degradation, accentuating the need for urgent conservation measures.     

Maternal immune system adaptation to pregnancy - a potential influence on the course of diabetic retinopathy

Background  

Progression of diabetic retinopathy occurs at least temporarily during pregnancy. Although the cause of this progression is not entirely understood, the immune phenomenon and chronic inflammation may play a significant role. During pregnancy in order to avoid fetus rejection, certain components of the immune system that are knowingly implicated in the pathogenesis of diabetic retinopathy are activated including generalized leukocyte activation and an increase in certain cytokine plasma levels. Activated leukocytes with up regulated adhesion molecules have an increased potential to bind to the endothelium cells of blood vessels. Leukocyte-endothelial interaction and the consequent leukostasis with capillary occlusion, ischemia and vascular leakage have a substantial role in the development of diabetic retinopathy. Furthermore, certain increased cytokines are known to cause blood-retinal-barrier breakdown whilst others promote angiogenic and fibrovascular proliferation and thereby can also be implicated in the pathogenesis of this diabetic complication.     

The occurrence and high diversity of Clostridium difficile genotypes in rivers

Clostridium difficile is mainly associated with nosocomial infections but can be present also in other environments. In this study we compared three methods (culturing with and without ethanol shock and real-time PCR) for detection of C. difficile in water and have used them on a series of river water samples. C. difficile was present in 17 of 25 rivers tested (68.0%) and in 42 of 69 water samples tested (60.9%). Positive sampling sites correlated with increased population densities. Isolates were distributed into 34 PCR ribotypes, of which more than half are present also in humans and animals. PCR ribotype 014 was the predominate type (16.2% of all isolates).     

Influence of sodium nitroprusside on human erythrocyte membrane water permeability: an NMR study

Sodium nitroprusside (SNP) is a nitric oxide (?NO) donor in vitro and in vivo. In this paper the time variation of the intracellular water proton nuclear magnetic resonance (NMR) effective relaxation time T'(2a) in SNP-treated human erythrocyte suspensions, containing 10 mM membrane impermeable paramagnetic MnCl2, has been measured. The observed T'(2a) time-course was analyzed in terms of the two mechanisms by which released ?NO affects T'(2a). These are, respectively, enhancement of the intracellular water proton intrinsic NMR relaxation rate 1/T(2a) by paramagnetism of ?NO subsequently bonded to iron atoms of intracellular deoxyhemoglobin, and suppression of diffusional water permeability P(d) as a consequence of nitrosylation of aquaporin-1 (AQP1) channel Cys189, either by direct reaction with ?NO or with one of the ?NO oxidation products, such as N2O3. The bound ?NO on the Cys189 thiol residue appears to impose a less efficient barrier to water permeation through AQP1 than the larger carboxyphenylmercuryl residue from p-chloromercuribenzoate. The effect of ?NO on P(d) is discussed in terms of NO-induced vasodilation.     

4,6-α-Glucanotransferase activity occurs more widespread in Lactobacillus strains and constitutes a separate GH70 subfamily

Family 70 glycoside hydrolase glucansucrase enzymes exclusively occur in lactic acid bacteria and synthesize a wide range of α-d-glucan (abbreviated as α-glucan) oligo- and polysaccharides. Of the 47 characterized GH70 enzymes, 46 use sucrose as glucose donor. A single GH70 enzyme was recently found to be inactive with sucrose and to utilize maltooligosaccharides [(1→4)-α-d-glucooligosaccharides] as glucose donor substrates for α-glucan synthesis, acting as a 4,6-α-glucanotransferase (4,6-αGT) enzyme. Here, we report the characterization of two further GH70 4,6-αGT enzymes, i.e., from Lactobacillus reuteri strains DSM 20016 and ML1, which use maltooligosaccharides as glucose donor. Both enzymes cleave α1→4 glycosidic linkages and add the released glucose moieties one by one to the non-reducing end of growing linear α-glucan chains via α1→6 glycosidic linkages (α1→4 to α1→6 transfer activity). In this way, they convert pure maltooligosaccharide substrates into linear α-glucan product mixtures with about 50% α1→6 glycosidic bonds (isomalto/maltooligosaccharides). These new α-glucan products may provide an exciting type of carbohydrate for the food industry. The results show that 4,6-αGTs occur more widespread in family GH70 and can be considered as a GH70 subfamily. Sequence analysis allowed identification of amino acid residues in acceptor substrate binding subsites +1 and +2, differing between GH70 GTF and 4,6-αGT enzymes.     

Screening of lactic acid bacteria from Indonesia reveals glucansucrase and fructansucrase genes in two different Weissella confusa strains from soya

Homopolysaccharide (glucan and fructan) synthesis from sucrose by sucrase enzymes in lactic acid bacteria (LAB) has been well studied in the genera Leuconostoc, Streptococcus and Lactobacillus . This study aimed to identify and characterize genes encoding glucansucrase/glucosyltransferase (GTF) and fructansucrases/fructosyltransferase (FTF) enzymes from genomic DNA of 'rare' Indonesian exopolysaccharide-producing LAB. From a total of 63 exopolysaccharide-producing LAB isolates obtained from foods, beverages and environmental samples, 18 isolates showing the most slimy and mucoid colony morphologies on sucrose were chosen for further study. By comparing bacterial growth on De Man, Rogosa and Sharpe (MRS)-sucrose with that on MRS-raffinose, and using the results of a previous PCR screening study with degenerate primer pairs targeting the conserved catalytic domain of GTFs, various strains were identified as producers of fructan (13), of glucan only (five) or as potential producers of both glucan and fructan (nine). Here, we report the characteristics of three gtf genes and one ftf gene obtained from Weissella confusa strains MBF8-1 and MBF8-2. Strain MBF8-1 harbored two putative gtf genes with high sequence similarity to GTFB of Lactobacillus reuteri 121 and GTF180 of L. reuteri 180, respectively. Strain MBF8-2 possessed single gtf and ftf genes with high sequence similarity to GTFKg3 of Lactobacillus fermentum Kg3 and DSRWC of Weissella cibaria , and FTF levansucrase of L. reuteri 121, respectively.